Mass Spectrometry - ACS Publications

Chapter 17

Anion Exchange Thermospray Tandem Mass Spectrometry of Polar Urinary Metabolites and Metabolic Conjugates 1

2

3

William M. Draper , F. Reber Brown , Robert Bethem , and Michael J. Miille 3

Downloaded by TUFTS UNIV on November 6, 2016 | http://pubs.acs.org Publication Date: February 22, 1990 | doi: 10.1021/bk-1990-0420.ch017

1

Hazardous Materials Laboratory, California Department of Health Services, 2151 Berkeley Way, Berkeley, CA 94704 California Public Health Foundation, 2151 Berkeley Way, Berkeley, CA 94704 ENSECO-California Analytical Laboratory, 2544 Industrial Boulevard, West Sacramento, CA 95691 2

3

Negative ion thermospray (TSP) tandem mass spectrometry (MS/MS) was used to determine phenols and their corresponding glucuronide and sulfate conjugates. Nine model compounds were separated to baseline on a strong anion exchange (SAX) L C column eluted with an ammonium formate buffer-acetonitrile mobile phase. Filament off TSP mass spectra provided molecular weight information for both glucuronide and sulfate conjugates (i.e., depronated molecule ions) as well as weak signals for sulfate hydrolysis products and dehydroglucuronic acid. TSP M S / M S provided much more structural information than TSP MS alone. Product ion spectra of the glucuronic acid conjugates revealed the aglycone mass as well as a "fingerprint" of glucuronic acid product ions. Under collisional activation the aryl sulfates fragmented efficiently to phenols detected as phenate anions. Selected reaction monitoring in L C / M S / M S allowed glucuronic- and sulfate -selective detection as well as specific detection of xenobiotics and their elaborated conjugates.

Foreign compounds absorbed by mammals are subject to a variety of metabolic processes including functionalization and conjugation, also known as Phase I and Phase II metabolism, respectively. Common Phase I transformations include oxidation, reduction, and hydrolysis while Phase II metabolism involves the biosynthesis of polar adducts (1). In general the metabolites of foreign compounds are more difficult to identify and quantitate than their parent structures due to their polarity and lower volatility. Metabolic Conjugation of Foreign Compounds in Mammals. Evidence for Phase II metabolism was first provided by Justus von Liebig who identified hippuric acid in equine urine in 1830 (2). Soon thereafter it was demonstrated by Ure and Keller that humans treated for gout with benzoic acid also excrete hippurate in the urine. These milestones mark not only the discovery of bioconjugation, but also the beginning of the scientific study of foreign compound metabolism in mammals. By 1900 the major metabolic conjugation pathways were known and included conjugation with glycine (as in the case of hippuric acid from benzoate), sulfate, glucuronic acid, ornithine and mercapturic acid. Methylation and acetylation of foreign compounds had been reported as well. The biochemistry of conjugation has been thoroughly reviewed in the recent publications of Caldwell (2), Hiron and Millburn Q), and a previous ACS Symposium Series monograph (4). Glucuronidation is the most versatile and quantitatively important metabolic pathway affecting a diverse group of substrates including alcohols, carboxylic acids, amines, and sulfur compounds (2.4). 0097-6156/90/0420-0253$06.00/0 © 1990 American Chemical Society Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

254

LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY


Glucuronyl transferases are membrane-bound enzymes primarily associated with the endoplasmic reticulum of liver cells. Glucuronidation takes place to a lesser extent in cells of many other tissues as well. Sulfate conjugation represents the important metabolic alternative to glucuronidation being mediated by a soluble enzyme system (2). Glucuronidation is favored for lipophilic compounds while the lower molecular weight, hydrophilic xenobiotics in the cytosol are preferred substrates for sulfation. Functionalizing and conjugating xenobiotics appears to play a role in their detoxification. Glucuronidation and sulfation usually lead to complete loss of pharmacological or pesticidal activity (5). The conjugates are highly water soluble, have reduced affinity for sensitive cellular receptors and are rapidly excreted via the kidney or bile. In rare cases Phase II bioconjugation appears to be involved in the activation of toxic compounds in the digestive and excretory organs (6). Xenobiotics thus are cleared from the body in a variety of forms: unchanged, as Phase I metabolites, as Phase II metabolites, or products of sequential Phase I and Phase II transformations. Conjugates are the predominant excreted form for most foreign compounds (2). Analytic Techniques for Determination of Metabolic Conjugates. The elucidation of metabolite structures was a remarkable achievement for chemists practicing before the Civil War. In spite of over 160 years of advancements in the chemical sciences, and the revolution in modern methods of instrumental analysis, structure elucidation and quantitative analysis of metabolic conjugates remain challenging. Gas-liquid chromatography (GC) or G C / M S has been used in the determination of glucuronides, but conversion to methylated, acetylated or trimethylsilylated volatile derivatives is required (2). Similarly, aryl sulfates are converted to volatile n-propyl derivatives prior to G C / M S analysis (£). Classical methods for glucuronide characterization involve enzymatic or chemical cleavage followed by identification of the aglycone (7). Similar approaches are used in sulfate conjugate identification. Modern desorption ionization MS methods including field, plasma and laser desorption MS, fast atom bombardment (FAB) and thermospray MS eliminate the requirement of sample volatilization (2). Unlike conventional MS techniques, analytes are transported to the ion source and ionized within the condensed phase. TSP MS in particular has been applied in the determination of glucuronides (10-16). and sulfates (16.17) as well as other polar metabolites and metabolic conjugates. Chemical Dosimetry bv TSP L C / M S . One of our long-term objectives in studying TSP L C / M S is the development of chemical dosimetry based on direct determination of polar metabolites in biological fluids. Most toxic substance exposure scenarios (i.e., near hazardous waste sites) involve complex and variable mixtures of substances. Biological monitoring, where human fluids, tissues and excreta are analyzed, measures actual exposure, whereas analysis of soil, air or water can only provide an estimate of potential exposure. Exposure data forms the basis of human health risk assessment, and ultimately defines cleanup requirements at contaminated sites. The mammalian liver through its tremendous metabolic flexibility disposes of many toxic substances, including those released from hazardous waste sites, in very few common, polar forms. In a sense we hope to exploit this capability to convert pollutants to polar, involatile forms amenable to direct TSP L C / M S determination. The development of improved means for separation, selective detection and identification of metabolic conjugates in biological fluids has applications in the dosimetry of many toxic substances. Such techniques may be useful in screening exposures to a multitude of compounds simultaneously. Goals of this Study. The purpose of this work is to evaluate the TSP tandem mass spectrometer as a detector for glucuronide and sulfate conjugates separated on a strong anion exchange L C column. Both TSP and product ion spectra were investigated using ammonium formate buffer ionization and negative ion detection. The impact of the TSP interface on chromatographic efficiency and the capabilities of selected reaction monitoring were evaluated. Experimental Chemicals. Sources and preparation of chemical standards are described elsewhere (16). Chromatography solvents were commercially available, pesticide residue grade and were ultrafiltered through a Teflon filter prior to use.

Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


17. DRAPER ETAL.

Anion Exchange Thermospray Tandem MS

255

Thermospray MS/MS. A Finnigan triple stage quadrupole (TSQ 70) instrument (San Jose, CA) equipped with a Finnigan TSP interface was operated routinely in negative ion mode with ammonium formate buffer ionization and filament off. Liberato and Yergey have referred to filament on ionization as "external" ionization, filament off ionization with or without a volatile buffer is designated "direct" thermospray ionization (IS). A Varian Model 5000 high pressure liquid chromatgraphy (HPLC) pump with double pulse dampeners was used and samples were introduced with a 20 uL loop injector. The mobile phase was 1.5 mL/min pH 4.5 0.05 molar ammonium formate-acetonitrile (3:2, v/v). Preliminary flow injection analysis (FIA) studies (no column) reported elsewhere (16) used an ammonium acetate-methanol mobile phase with the filament on and both negative and positive ion detection. Mass spectrometer settings were typically: ion source temp., 230°C; TSP aerosol temp., 265°C; vaporizer temp., 126°C. For the initial acquisition of full scan spectra, the instrument was scanned from m/z 60 to m/z 600 in 1 s. The repeller was set to 47 volts and lens settings were optimized by daily tuning with adenosine (m/z 136, m/z 268) or a polypropylene glycol mixture (acetate adduct ions at m/z 367, m/z 425, and m/z 483) or 4-chlorobenzenesulfonic acid (m/z 191 and 193) using the ammonium formate or ammonium acetate mobile phase and flow rate described above. Instrument tuning, acquisition, and data processing were carried out with Finnigan's Instrument Control Language (ICL) and ICIS data system. Collision activated dissociation (CAD) studies used argon as a collision gas at a pressure of approximately 1.4 mtorr. Collision energies were adjusted between -9V and -40V with a goal of reducing the precursor ion intensity to less than 20% of the base peak in the product spectrum. Strong Anion Exchange H P L C Separation. The strong anion exchange (SAX) L C separation of phenols and glucuronide and sulfate conjugates has been reported elsewhere (19). Briefly, a 5 um Supelco 4.6 mm X 25 cm LC-SAX column (Bellefonte, PA) is eluted isocratically with the ammonium formate-acetonitrile mobile phase described above. A precolumn packed with the same stationary phase and a particle filter were used. Ammonium acetate could be substituted for the formate buffer, but acetonitrile was the only organic modifier successfully used. The chromatographic conditions were optimized using an absorbance detector monitoring at 254 nm. The S A X column performance was maintained by washing the column with a phosphate buffer after use, and the column also was stored in phosphate buffer (19). As shown in Figure 1, phenols elute prior to glucuronides which are in turn followed by the sulfate conjugates. The mobile phase chosen was particularly advantageous for TSP and avoided postcolumn addition of a volatile TSP ionization buffer (20). Results and Discussion Negative Ion TSP Mass Spectra. Thermospray spectra for phenols revealed only deprotonated molecule ions (ArO") and formate adduct ions ([M + HCOO]") (Table I). 4-Nitrophenol, the strongest acid in solution, and presumably the gas phase, favored formation of ArO" while phenol exhibited only the adduct ion. A deprotonated dimer anion ([2M - H])~ was detected in the case of 4-nitrophenol. The spectra were similar with the LC-SAX column in line. The minor changes in ion relative intensity when FIA spectra were compared to L C / M S spectra are not uncommon in TSP mass spectrometry. TSP spectra are concentration dependent and with the column in line detector concentration is reduced due to band broadening. Thermospray mass spectrometry exhibits limited day-to-day reproducibility of ionization efficiency and fragmentation patterns, and a dependence of ion intensity on flow rate (21). TSP spectra also are affected by pressure, temperature, and vapor composition (22.23) and apparently also the design of the TSP source (24). Glucuronide mass spectra also provided molecular weight information, in this case the deprotonated molecular anions were base ions for phenyl-beta-D-glucuronide and 1-naphthyl-beta-D-glucuronide (Table II). Solvolysis (or pyrolysis) was very important for 4nitrophenyl-beta-D-glucuronide where the aglycone deprotonated molecule ion was most abundant and its formate adduct was also present. The formate adduct ion of dehydroglucuronic acid, m/z 221, was diagnostic for glucuronic acid conjugates under the thermospray conditions studied. Other glucuronic acid-derived ions including m/z 193 and m/z 175 were detected sporadically in low abundance. Withfilamentexternal ionization, molecular anions (M") were observed for glucuonide structures (16) indicating ionization by resonance electron capture. Direct thermospray ionization occurs only by proton transfer or ion attachment.


256

LIQUID

CHROMATOGRAPHY/MASS

SPECTROMETRY


COO"

Time (minutes) Figure 1. Strong anion exchange L C separation of phenols, aryl glucuronides and aryl sulfates using a U V absorbance detector. Compounds eluted are: 1, phenol; 2, 4 - n i t r o p h e n o l ; 3, 1-naphthol; 4, p h e n y l - b e t a - D - g l u c u r o n i d e ; 5, 4-nitrophenyl-beta-D-glucuronide; 6, 1- naphthyl-beta-D-glucuronide; 7, phenyl sulfate; 8, 4-nitrophenyl sulfate; 9, 1-naphthyl sulfate. (Reproduced with permission from Ref. 19. Copyright 1989 Elsevier Science Publishers B.V.)



b

b

b

m/z 189(100) m/z 189(63) m/z 184(14) m/z 184(32)

m/z 143(100) m/z 138(100) m/z 138(100)

144 139 139

m/z 140(100)

[M + HC00]"

m/z 143(16)

-

ArO"

144

94

MW

m/z 277(32)

m/z 277(13)

[2M - H]~

b HPLC-MS s p e c t r a recorded w i t h s t r o n g anion exchange column i n l i n e and the same mobile phase.

a Flow i n j e c t i o n (no column) s p e c t r a were recorded w i t h f i l a m e n t o f f and ammonium formate b u f f e r - a c e t o n i t r i l e u n l e s s otherwise i n d i c a t e d . The s c a n r a n g e f o r p h e n o l was m/z 50 t o m/z 150 and m/z 50 t o m/z 400 f o r 4-nitrophenol and 1-naphthol

4-nitrophenol

4-ni tropheno1

l-naphthol

1-naphthol

phenol

Compound

Ions ( R e l a t i v e I n t e n s i t y , %)

a

Table I. Thermospray Mass S p e c t r a of Phenols i n Ammonium F o r m a t e - A c e t o n i t r i l e



b

a b c d e f g

6

8

6

D

315

See footnote a i n Table 1. See footnote b i n Table 1. [M - C H 0 - H]~ [ g l u c u r o n i c a c i d - H]~ [dehydroglucuronic a c i d - H]~ [ArOH + HC00]~ [M - H - N0]~

4-nitrophenyl-b-D-glucuronide

4-nitrophenyl-b-D-glucuronlde

b

320

270

MW 6

Q

[C H 0 6

+ HCOO]"

-

314(92)

m/z 314(4.8)

m/z

221(27)

m/z 221(19)

m/z

m/z 319(100) m/z 221(33)

m/z 319(100) m/z 221(14)

m/z 269(100)

m/z 269(100) m/z 221(5.3)

[M - H ] "

Ions ( R e l a t i v e I n t e n s i t y , *)

138(100)

143 (5.3)

m/z 138(100)

m/z

m/z

ArO"

d

a

e

m/z m/z 175(8.0) , m/z

e

m/z 175(7.6) , m/z 284(20)9

184(24)

f

f

184(23) ,

m/z 135(18). m/z 165(7.6), m/z 1 9 3 ( 6 . l ) , m/z 259(4.6), m/z 328(4.6), m/z 387(12)

m/z 141 (20)

Other Ions

Thermospray Mass Spectra of A r y l Glucuronides In Ammonium F o r m a t e - A c e t o n i t r i l e

l-naphthyl-b-D-glucuronide

1-naphthyl-b-D-glucuronide

phenyl-b-D-glucuronide

phenyl-b-D-glucuronide

Compound

Table I I .


17.

DRAPER E T A L .


259


Deprotonated molecule ions were the base peaks for phenol- and napththol sulfates. In the case of 4-nitrophenol sulfate, however, the relative intensity of the [M - H]" ion was much lower (Table III). Electron-withdrawing substituents, like the jj-nitro function, promote hydrolysis of sulfates, possibly explaining the susceptibility of 4- nitrophenyl sulfate to solvolysis in TSP MS. In previous external ionization TSP studies with the same source block temperature and a pH 4.5 ammonium acetate-methanol mobile phase, solvolysis of the sulfate conjugates was more prevalent (16). In fact, molecular species were not detected under these conditions. Low source block temperatures are important for obtaining molecular weight information in TSP MS (25), but this parameter was not responsible for the observed solvolysis of the aryl sulfates in this study. We propose that the cleavage of aryl sulfates in filament external ionization TSP (16) results from dissociative e" capture. Electron capture chemical ionization occurs only with filament or discharge electrode external ionization. A weak signal for the deprotonated dimer was also observed for phenol sulfate in the present study. TSP L C / M S . Negative ion L C / M S was carried out with the SAX column in line. Single ion monitoring (SIM) traces for the conjugate deprotonated molecule ions are plotted in Figure 2. The average chromatographic efficiency for the anion exchange L C / M S separation was -6,000 theoretical plates. Using the same column, precolumn, mobile phase and flow rate, a chromatograph equipped with an absorbance detector had higher efficiency (Table IV). For the glucuronide and sulfate conjugates the average loss in chromatographic efficiency (N) was 40% when using the TSP interface. These data demonstrate that the TSP interface, vacuum system, and ion source contribute to band broadening. While this did not effect the resolution of compounds with high capacity factors (k'), it hindered resolution of early eluting compounds like the free phenols. The total ion current (TIC) chromatogram where m/z 139 (phenol [M + HCOO]'), m/z 138 (4-nitrophenol [M - H]"), and m/z 189 (1-naphthol [M + HCOO]") were monitored showed a single, broad peak at 2.58 min. In contrast, baseline separation for the phenols was accomplished with an absorbance detector (Figure 1). The band broadening associated with the TSP system emphasizes the importance of chromatographic retention and resolution of analytes for optimum specificity, particularly in conventional TSP L C / M S , and to a lesser degree in TSP L C / M S / M S . The SIM traces plotted in Figure 2, representing 2 ug of each compound, show very little baseline noise. Instrument detection limits for these conjugates are expected to be well below 100 ng. Product Ion Spectra. The phenols resist collisionally activated dissociation (CAD) fragmentation, even at high collision energies. In contrast formate (Table V) or acetate adduct ions (in ammonium acetate TSP) and deprotonated dimer ions dissociate, even at very low activation energies. In each case the phenate ions are base peaks in the product ion spectrum. Among the phenols, only the 4-nitrophenol [M - H]" precursor ion underwent C A D fragmentation by loss of NO (Table III) and both NO and N 0 at higher energies (1£). The sulfates as a class were fragmented by a single pathway, neutral loss of 80 mass units corresponding to loss of SO,. Consistent with its tendency toward dissociative electron capture (or solvolysis) in filament-on T$P (16) and hydrolysis, 4-nitrophenol sulfate was most susceptible to S 0 loss in C A D fragmentation (Table V). With a collision energy of only 14 eV, the parent ion was either absent or only a minor product ion. Product ion spectra for glucuronide conjugates are rich in structural information in comparison to the sulfates. Diagnostic glucuronide product ions include m/z 59, m/z 85, m/z 113, and m/z 175 (Table VI) and Ref 1£. The m/z 113 glucuronide-derived product anion is always prominent. Positive product ion spectra of the ammoniated glucuronide adduct exhibit a fingerprint cluster of ions as well, consisting of m/z 113, m/z 159 and m/z 193 or m/z 194 (1£). The aglycone depronated molecule anion is a prominent product ion at low collision energies, and with increasing collision energies glucuronide-derived ions diminish leaving only the aglycone signal. In a previous study of glucuronide C A D mass spectra, 20 ug samples were introduced by flow injection (1£). The diagnostic spectral features, however, are also detected with 2 ug samples in L C / M S spectra where band broadening further lowers detector concentration. Most of the fingerprint product ions originate solely from C A D fragmentation except for the ubiquitous dehydroglucuronic acid deprotonated molecule ion, m/z 175. This ion is observed in TSP spectra, particularly in cases like 4-nitrophenyl-beta-D-glucuronide where facile solvolysis occurs. The conjugate cleavage mechanism occurring both in the thermospray source and the 2

3


260

LIQUID CHROMATOGRAPHY/MASS

SPECTROMETRY


CO UI'



m/z

314

COO"

50-

% 100-

50-1

* 100- m / z

5:18 2342

100

160023

150

HOV ^ ~

)—o

150

200

300

A

15:01 49258

350

400

250

300

350

400

T—1—1—1—1—1—(—1—1—1—1—|—1—1—1—1—1—1—r-

2.50

450

450

3

oso "

Figure 2. TSP SIM traces for metabolic conjugates eluted from a strong anion exchange column. Deprotonated molecule ions are plotted as follows: m/z 173, phenyl sulfate; m/z 269, phenyl-beta-D- glucuronide; m/z 218, 4-nitrophenyl-beta-D-glucuronide; m/z 314, 4nitrophenyl-beta-D-glucuronide; m/z 223, 1-naphthyl sulfate; m / z 319, 1-naphthyl-beta-D-glucuronide.

100

CCO"

200

-1—j—1—1—»—1—j—r—T—1—r-*)—r~i—1—1—p

m / z 319

223

50

0" -t—r~r'-i—|—t—t—r—Y—pn—i—i—r—j-—i—r—i—i—j—i—i—i—i—|—i—i—i—i—|—r—;—r—i—|—i—r—i—?—j—i—i—i—i—j—r~

20-

40-

60-

89-

lee-

5: 15

42420


sE+04 " l . 526

*E+03 3.026

4.616

*E+03


3

223

1-naphthol s u l f a t e , K+ s a l t m/z 189(7.6) m/z 189(2.4)

m/z 223(100)

m/z 184(33)

m/z 184(13)

[ArOH + HCOO]"

m/z 223(100)

m/z 218(42)

m/z 173(100)

ArOSO.

m/z 138(100) m/z

d

c

(3:2, v/v)

277(4)

d

m/z 347(2.5)

Other Ions

m/z 138(100) m/z 277(21)

3

Flow i n j e c t i o n (no column) w i t h 1.5 mL/min of 0.05 molar ammonium f o r m a t e - a c e t o n i t r i l e Anion mass [2 A r 0 S 0 + H]~ [2 ArOH - H]~ HPLC MS spectrum w i t h s t r o n g anion exchange column i n l i n e

218

4-nitrophenol s u l f a t e , K+ s a l t

a b c d e

173

phenol s u l f a t e , K+ s a l t

C

MW

Compound

ArO"

Thermospray Mass Spectra of A r y l S u l f a t e s

Ions ( R e l a t i v e I n t e n s i t y , %)

Table I I I .



(min)

19.5

13.4

233

218

16, 000 12, 000

173

17, 000

319

R

314

8, 300 5, 600

269

7,100

Ion (m/z)

15 .0

10 .4

9 .08

7 .27

5 . 15

5..00

(min)

TSP/LC/MS

7,500

4,300

12,000

7,000

2,700

2,900

N

2

a N = chromatographic e f f i c i e n c y c a l c u l a t e d from peak width a t h a l f height using the f o l l o w i n g equation: N = 5.54 ( t / v*i/2 )

1-naphthyl s u l f a t e

4-nitrophenyl

sulfate

11.0

sulfate

phenyl

5.79

5.37

R

7.15

t

1-naphthyl glucuronide

glucuronide

glucuronide

4-nitrophenyl

phenyl

Compound

LC/UV

Table IV. Chromatographic E f f i c i e n c y Determined f o r LC/UV and TSP/LC/MS Separation of Glucuronides and S u l f a t e s on a Strong Anion Exchange Column



[M - H]" [M - H]~ [M - H]~ [M - H]~

4-nitrophenol

phenol s u l f a t e

1-naphthyl s u l f a t e

4-nitrophenyl

b

a

[M + HCOO]"

1-naphthol

3

3

3

HCOO"" [M - H ] " [M + HCOO]*" [M - H - NO]~ [M - HI" [M - H - S 0 ] " [M - H]" [M - H - S 0 ] " [M - H]" [M - H - S 0 ] " [M - H]"*

Proposed Ion 45 143 189 108 138 93 173 143 223 138 218

m/z

a

b

93,314 --> 138, and 319 --> 143 neutral losses, respectively (Figure 3). The tandem mass spectrometer also can operate as a relatively non specific detector by, for example, monitoring the neutral loss of 46 mass units (HCOOH) when using ammonium formate buffer ionization or 60 mass units (CH COOH) when using acetate buffer ionization. In Figure 3, naphthol is detected in such a manner by monitoring the 189 --> 143 neutral loss. S R M provides additional selectivity in detecting an individual xenobiotic and its related metabolites. A moiety or substructure in the xenobiotic is tagged by its novel C A D fragmentation. In the case of 4-nitrophenol, the neutral loss of 30 mass units (NO) labels the nitrophenyl moiety (actually the N O substituent). With Q- locked at m/z 138 and Q locked at m/z 108 we detect 4-nitrophenol and its glucuronide and sulfate conjugates (Figure 3). Presumably, other metabolite structures in which the nitrophenol moiety is retained would be detected similarly. In practical application scanning can be manipulated on-the-fly within a chromatographic separation to obtain maximum information. In metabolism studies or as a chemical dosimeter, the structural feature of the parent compound and its unique neutral loss occurring on collisional activation, marks the metabolite. The mass of the metabolite is then obtained from the TSP mass spectrum at Q and the product ion spectrum of the metabolite molecule ion is obtained by product ion scanning. Recent publications have discussed additional applications of both tandem mass spectrometry (26. 27) and thermospray tandem mass spectrometry (28) in metabolite structure elucidation. 3

1

3

1

3

3

?

3

1

Conclusion As a soft ionization technique thermospray mass spectrometry often provides little fragmentation of molecular species. More importantly from the viewpoint of a metabolism chemist, thermospray accomplishes desorption ionization of extremely low vapor pressure analytes including intact glucuronide and sulfate conjugates. As demonstrated in the chapter by Brown and Draper in this proceedings, particle beam mass spectrometry does not have this capability.



0

50

10U

50

100

50

100

50

100

59

-

1 00

RIC

2:00

-i—«—j—•

4:00

• — i

Ite

1

f

3

"t« *

133

132

1

6:00

1

185

-•-•rn^wn

186

3I9~-*I43~

I38~-H08~

1

229 8:00

1

1^

r

' "I

269

\ " \

26? i

'

218~-*138~

269

10:00

rr-

A

173~-*93~

i— —i— —j— —r—'

314^138*

'2

125

'

1

'

376 i

3

*E+03 4.975

9.996

*E+04

4.574

"

379

Vmrm

1.036

*E+05

223-+143*" 9.110

14:00

1

nr-r-« • 1—.

12:00

1

,

Figure 3. Selected reaction monitoring of aryl glucuronides and sulfates eluted from a strong anion exchange column. Glucuronides are detected by neutral loss of 176 mass units as in phenyl-beta-D- glucuronide (269" --> 93"), 4-nitrophenyl-beta-D-glucuronide (314* --> 138"), and 1-naphthyl-beta-D-glucuronide (319" --> 143"). Sulfate conjugates are detected by neutral loss of S 0 as in phenyl sulfate (173" - > 93"), 4-nitrophenyl sulfate (218" --> 138") and 1-naphthyl sulfate (223" --> 143"). Compounds with a 4-nitrophenol moiety are detected with Q at m/z 138 and Q at m/z 108: scan 74, 4-nitrophenol; scan 133, 4-nitrophenyl-beta-D-glucuronide; and scan 269, 4- nitrophenyl sulfate.

i

A..

189"* 143" 79

A.

74

A

269-* 93



268

LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY

Separation of metabolite conjugates with the strong anion exchange H P L C column is extremely well suited to thermospray mass spectrometry. This L C / M S application has not been previously reported in the literature. The ammonium formate/acetonitrile or ammonium acetate/acetonitrile mobile phases supply the volatile ions required for TSP ionization. The L C / U V separation was readily adapted to TSP L C / M S with some loss in chromatographic efficiently. The band broadening encountered restricted the resolution of low k' compounds, but was inconsequential in the baseline separation of the the aryl glucuronide and aryl sulfate compounds studied. Losses in efficiency observed in TSP L C / M S stress the importance of chromatographic retention and phase selectivity in obtaining high quality, interference free L C / M S spectra. Product ion spectra provide a fingerprint of diagnostic ions as well as the aglycone mass for glucuronide conjugates. The prominence of these two spectral features can be controlled by the collision energy. The sulfate conjugates fragment by a single major process in the collision chamber, neutral loss of SO~. Through selected reaction monitoring the tandem mass spectrometer affords glucuronide- or sulfate specificity, a capability of obvious importance in the elucidation of metabolite structures. The tandem mass spectrometer's ability to selectively detect substructures of a xenobiotic (i.e., the nitrophenate moiety by the neutral loss of 30 mass units from m/z 138) allows selectively not unlike that afforded by radiolabels. Through the application of desorption ionization L C / M S and tandem MS technologies, the determination and identification of polar metabolites and metabolic conjugates may finally become routine, 160 years after the work of von Liebig! Acknowledgments The work was performed in part at the California Department of Health Services Hazardous Materials Laboratory. This study was supported in part by the Superfund Program Project No. 04705 from the National Institute of Environmental Health Sciences of the National Institutes of Health. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Doull, J.; Klassen, C.; Amdur, M . , Eds. Toxicology, The Basic Science of Poisons; Academic Press: New York, 1966. Caldwell, J. In Xenobiotic Conjugation Chemistry; Paulson, G.; Caldwell, J.; Hutson, D.; Menn, J., Eds.; ACS Symposium Series No. 299; American Chemical Society: Washington, D C 1986; pp 2-28. Hiron, P. C.; Millburn, P. In Foreign Compound Metabolism in Mammals, Volume 5; Hathway, D. E., Ed.; The Chemical Society: London, 1979; pp 132-158. Paulson, G.; Caldwell, J.; Hutson, D.; Menn, J., Eds. Xenobiotic Conjugation Chemistry; ACS Symposium Series No. 299; American Chemical Society: Washington, DC, 1986. Crawford, M. J.; Hutson, D. H. In Bound and Conjugated Pesticide Residues; Kaufman, D.; Still, G.; Paulson, G.; Bandal, S., Eds.; ACS Symposium Series No. 29; American Chemical Society: Washington, DC, 1979; pp 181-229. Snyder, R.; Parke, D . V.; Kocsis, J. J.; Jollow, D. J.; Gibson, C. G.; Witmer, C. M . , Eds. Biological Reactive Intermediates; Plenum Press: New York, 1982. Bakke, J. E . In Bound and Conjugated Pesticide Residues; Kaufman, D.; Still, G.; Paulson, G.; Bandal, S., Eds.; ACS Symposium Series No. 29; American Chemical Society: Washington, DC, 1979; pp 55-67. Paulson, G.; Simpson, M . ; Giddings, J.; Bakke, J.; Stolzenberg, . Biomed. Mass Spectrom. 1978, 5, 413. Fenselau, C.; Yellet, L . In Xenobiotic Conjugation Chemistry; Paulson, G.; Caldwell, J.; Hutson, D.; Menn, J., Eds.; ACS Symposium Series No. 299; American Chemical Society: Washington, DC, 1986; pp 159-176. Liberato, D . J.; Fenselau, C. C.; Vestal, M. L.; Yergey, A. L. Anal. Chem. 1983, 55, 1741. Watson, D.; Taylor, G . W.; Murray, S. Biomed. Environ. Mass Spectrom. 1986, 13, 65. Betowski, L. D.; Korfmacher, W. A.; Lay, J. O., Jr.; Potter, D. W.; Hinson, J. A . Biomed. Environ. Mass Spectrom. 1987, 14, 705. Blake, T. J. A . J. of Chromatogr. 1987, 394, 171. Korfmacher, W. A.; Holder, C. L.; Betowski, L. D.; Mitchum, R. K. J. Anal. Toxicol. 1987, 11, 182.


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